The correct patterning and timing of the fusion of cranial sutures is essential for normal skull development. Defects in suture formation lead to malformations and deformations, which cause constriction of the cranium and can impair cognition. Our knowledge of the mechanistic bases that distinguish normal from abnormal suture development is incomplete. The zebrafish is a powerful genetic and experimental model for exploring fundamental processes in vertebrate skeletal development. We have performed forward genetic screens in zebrafish to identify adult fish with defects in suture formation that are relevant to defects occurring in human patients. To efficiently map and clone the responsible genes, we developed new massively parallel sequencing and analytic strategies. This enabled us to identify bone morphogenetic protein 1a (bmp1a) and integrin 10 (itg10) as important participants during suture formation. Identifying genes involved in the post- translational modification of extracellular matrix (ECM) proteins and in the signaling that occurs between the ECM and the cell suggests changes in ECM and ECM-regulated cell signaling may comprise a shared downstream pathway for many genetic and environmental causes of abnormal suture development/closure. The goal of this proposal is to investigate the role of ECM post-translational modification and, specifically, the role of Bmp1 protease during normal and pathological suture formation. We will achieve these goals by completing 3 Aims. In Aim 1 we will identify new genetic regulators of suture formation by using mutants deficient in bmp1a to screen for dominant modifiers of Bmp1a function. Our preliminary data indicate this is a highly productive strategy. In Aim 2 we will characterize the effects of impaired Bmp1 activity on suture ECM proteins in zebrafish and determine whether altered modification of ECM proteins also occurs in synostoses from human patients. We have access to surgically excised affected and adjacent normal suture tissue from patients with known syndromic and unknown causes of synostosis. In Aim 3 we will capitalize on the zebrafish model's ability to be effectively utilizedin small molecule screens. We will specifically use bmp1a mutant fish to identify small molecules that alter Bmp1 activity in vivo. Molecules that affect the earliest phenotype observed in bmp1a mutant fish (altered finfolds) can then be tested for therapeutic efficacy in the late onset-phenotype (synostosis) using localized chemical- impregnated microbead implantation assays. The strength of the zebrafish model is that it enables the efficient identification of genes and pathways important for suture formation using forward genetic methods. Genes found to affect sutures in the zebrafish can then be studied in mammalian models of synostosis. Zebrafish are also useful for high-throughput in vivo screens for small molecule compounds that can inhibit or potentiate suture fusion. Compounds that alter deleterious phenotypes in zebrafish with Bmp1a mutations may have therapeutic value in patients with synostosis or with BMP1-associated phenotypes.